Second harmonic generation (SHG) is a nonlinear optical process which may be configured as a surface-selective detection technique that enables detection of binding interactions and conformational change in proteins and other biological targets using second harmonic-active labels attached to the target molecules (see, for example, U.S. Pat. Nos. 6,953,694, and 8,497,073). To date these methods have been applied to detect ligand-induced conformational changes in a variety of systems and to distinguish ligands by the type of conformation they induce upon binding (Salafsky, J. S. (2001), “‘SHG-labels’ for Detection of Molecules by Second Harmonic Generation”, Chemical Physics Letters 342, 485-491; Salafsky, J. S. (2003), “Second-Harmonic Generation as a Probe of Conformational Change in Molecules”, Chemical Physics Letters 381, 705-709; Salafsky, J. S. (2006), “Detection of Protein Conformational Change by Optical Second-Harmonic Generation”, Journal of Chemical Physics 125; Moree, B., et al. (2015), “Small Molecules Detected by Second Harmonic Generation Modulate the Conformation of Monomeric α-Synuclein and Reuce Its Aggregation in Cells”, J. boil. Chem. 290(46); 27582-27593; Moree, et al. (2015), “Protein Conformational Changes are Detected and Resolved Site Specifically by Second-Harmonic Generation”, Biophys. J. 109:806-815). Examples include distinguishing between type I vs. type II kinase inhibitors, such as imatinib and dasatinib, which bind to the protein to induce inactive and active conformations, respectively.
SHG and the related technique sum-frequency generation (SFG) have been used in the past to study the orientation of dye molecules at an interface (Heinz T., et al., (1983), “Determination of Molecular Orientation of Monolayer Adsorbates by Optical Second-Harmonic Generation”, Physical Review A 28(3):1883-1885; Heinz, T, (1991) Second-Order Nonlinear Optical Effects at Surfaces and Interfaces”, in Nonlinear Surface Electromagnetic Phenomena (Stegeman, H. P. a. G. ed.), Elsevier, Amsterdam, pp 353-416). In these measurements, the components of the nonlinear susceptibility (χ(2)) of the labeled interface are determined using polarized light. Details of the molecular orientation distribution for the dye molecules at the interface can then be inferred using the experimentally determined values for χ(2) and assumptions regarding the degree of orientation of the dye molecules within the plane of the interface, the relative magnitude of the components of hyperpolarizability (α(2)) of the dye molecules in the molecular frame of reference, etc.
The field of protein and biomolecular structure determination is highly developed but there remains a need for a sensitive and rapid measure of conformational change and structure in real time and in solution. Most information about protein structure and dynamics has come mainly from X-ray crystallography and NMR studies, but these techniques are relatively labor and material intensive, slow, or provide only a static snapshot of protein structure.
The presently disclosed methods, devices, and systems for determining protein structure using surface-selective nonlinear optical techniques address these unmet needs. In some embodiments, determination of protein structure in a high-throughput format is enabled through the use of novel device designs and mechanisms for rapid, precise, and interchangeable positioning of substrates (comprising the tethered or immobilized biological targets to be analyzed) with respect to the optical system used to deliver excitation light, and which at the same time ensure that efficient optical coupling between the excitation light and the substrate surface is maintained. One preferred format for high-throughput optical interrogation of biological samples is the glass-bottomed microwell plate. The systems and methods disclosed herein provide mechanisms for coupling the high intensity excitation light required for SHG and other nonlinear optical techniques to a substrate, e.g. the glass substrate in a glass-bottomed microwell plate, by means of total internal reflection in a manner that is compatible with the requirements for a high-throughput analysis system.